Molecular characterization of Argulus bengalensis and Argulus siamensis (Crustacea: Argulidae) infecting the cultured carps in West Bengal, India using 18S rRNA gene sequences.

The present study characterized Argulus spp. infecting the cultured carps using 18S rRNA gene sequences, estimated the genetic similarity among Argulus spp. and established their phylogenetic relationship. Of the 320 fish samples screened, 34 fish (10.6%) had Argulus infection. The parasitic frequency index (PFI) was observed to be high (20%) in Hypophthalmichthys molitrix and Labeo bata. The frequency of infection was high in September (PFI: 17%) and October (PFI: 12.9%). The 18S rRNA sequences of five A. bengalensis (KF583878, KF192316, KM016968, KM016969, and KM016970) and one A. siamensis (KF583879) of this study showed genetic heterogeneity and exhibited 77-99% homology among the 18S rRNA gene sequences of Argulus spp. of NCBI GenBank database. Among the Indian Argulus spp. the sequence homology was 87-100%. Evolutionary pair-wise distances between Indian Argulus spp. and other Argulus spp. ranged from 0 to 20.20%. In the phylogenetic tree, all the crustaceans were clustered together as a separate clade with two distinct lineages. The lineage-1 comprised exclusive of Branchiura (Argulus spp.). All Argulus bengalensis clustered together and A. siamensis (KF583879) was closely related to Argulus sp. JN558648. The results of the present study provided baseline data for future work on population structure analysis of Indian Argulus species.


INTRODUCTION
Various infectious and non-infectious diseases are reportedly affecting the cultured fish species, resulting in heavy economic loss to global aquaculture industry [1,2]. The outer surfaces of the fish are exploited by a huge diversity of parasites including protozoans, monogeneans, leeches, molluscs, crustaceans, etc. The genus Argulus (fish louse) of the family Argulidae (fish lice) is one of the most damaging ectoparasites of fish and is of major economic concern in all phases of the aquaculture industry from production to marketing [3]. Argulus spp. are the cosmopolitan fish parasites commonly known as fish lice and are distributed throughout the world except Antarctica [3]. These parasites may survive on the freshwater and marine fish species as hosts for most part of their life cycle. It can also survive in variable range of temperatures and its intensity is higher in culture conditions than in natural environment [2][3][4]. About 129 species of Argulus with a complex life cycle involving several metamorphic stages have been reported [3]. Argulus spp. cause skin lesions on the host by their suckers and proboscis while feeding, which often lead to secondary infections by bacteria and fungi. Epidemics of argulosis have been reported in carp and trout by producing severe damage to fish stocks [2]. Argulosis cause weight loss by reducing appetite and anaemia in fish. Acute infection of Argulus spp. results in fish loss due to direct effects such as dermal ulceration, osmotic imbalance, physiological stress, and immunosuppression [5]. In India, the total loss due to argulosis in carp culture has been estimated to the magnitude of Rs. 29,524.40 /ha/year (1 USD = Rs. 68) [6].
Identification and characterization of Argulus has assumed prime importance so that suitable preventive and control measures could be taken up to reduce fish mortality and production loss. Various fingerprinting methods can be broadly classified and genome based approaches are now in use to identify parasites. Genetic data and phylogeographical structure of a species provide the basic information required to understand their evolution and biogeographical history. The direct sequencing of rRNA gene is generally accepted as a stable and specific marker for identification [7]. Classification of parasites through this technique may be an effectively useful for phylogenetic identification at various levels. Studies on the genetic diversity of Argulus spp. and phylogenetic relationship are very limited [8,9]. A comparative phylogenetic analysis between Pentastomida cytochrome C oxidase subunit1 (pcox1), cox2 and NADH dehydrogenase subunits (pnad4) of A. americanus was reported [10]. Recently, the genetic diversity of Argulus spp. from major aquaculture zones in India by RAPD analysis has been described [11]. Data pertaining to molecular characterization and phylogenetic relationship of Indian Argulus spp. are, however, not available. The present study was, therefore, designed to characterize the Argulus spp. infecting the cultured carps of India by molecular tools, to estimate genetic similarity within and among Argulus populations and to establish phylogenetic relationship.  (Table 1) for determining the prevalence of Argulus infection. A total of 15 ponds were surveyed from five locations and all the fish were brought to the laboratory within 4 h of collection in oxygen filled polythene bags. Each live fish was examined thoroughly in the laboratory for the presence of Argulus as described by Natarajan [4]. Argulus spp. from infected carps were isolated carefully and transferred on to clean grease free glass slides with few drops of distilled water, covered with cover slips and sealed with Distrene, Plasticizer and Xylene (DPX). The slides were examined under 4X lens of upright microscope (Motic BA400) and subsequently under stereomicroscope (Olympus SZ 40). The parasite was identified based on the morphometric criteria [12].

Determination of parasitic frequency index (PFI) and severity of infection:
The parasitic frequency index (PFI) was calculated by taking the number of hosts infected by Argulus against the total number of hosts examined. The severity of infection was determined by following the scale proposed by Lightner [13] with slight modification: 0 = no signs of parasite, 0.5 = a very few scattered signs of parasitic infection; 1 = low parasitic infection; 2 = low to moderate parasitic infection, 3 = moderate parasitic infection, 4 = severe parasitic infection.

Molecular analysis:
For molecular characterization, two morphologically distinct Argulus species, viz., A. bengalensis from L. rohita (n=5) and A. siamensis from C. carpio (n=1) were preserved in 70% ethanol and stored at 4ºC until extraction. The DNA was extracted according to protocol described in Anjan et al. [14]. The 18S small subunit ribosomal RNA (18S rRNA) was amplified by Gradient PCR system (Eppendorf Master cycler Pro S) using a set of universal eukaryotic primers -ERIB1, 5´-ACC TGG TTG ATC CTG CCA G-3´ and ERIB10, 5´-CTT CCG CAG GTT CAC CTA CGG-3´ [15]. A 25 μL volume of PCR-mixture prepared with 20 mmol/L Tris-HCl (pH=8.4), 50 mmol/L KCl, 2.0 mmol/L MgCl2, 200 umol/L of dNTPs, 1 umol/L of each primer, 30 ng of genomic DNA and 1.5 U of Taq DNA polymerase. Amplification was done by initial denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 30 sec, annealing of primers at 51°C for 30 sec and extension at 72°C for 60 sec. The final extension was at 72°C for 5 min. The PCR amplicons were analysed on a 1.5% agarose gel containing 0.5 μg/mL ethidium bromide in 1X Trisacetate-EDTA (TAE) buffer. The DNA bands were visualized and documented using a Gel documentation system (Syngene G-Box F3). The amplified PCR product was first MBRC http://mbrc.shirazu.ac.ir 159 purified using EXO-SAP treatment. The concentration of the purified DNA was determined and subjected to automated DNA sequencing on ABI 3730xl Genetic Analyzer (Applied Biosystems, USA). Sequencing was carried out using BigDye® Terminator v3.1 Cycle sequencing kit (Applied Biosystems, USA) following manufacturers' instructions. Chromatogram of sequence data confirmed the peak, reproducibility, quality of gene sequences. Raw sequence data was arranged as complementary and consensus through online Reverse-Complement tools and Genefisher2. Multiple sequence alignments (MSA) were carried out in 3 states as pairwise alignments by ClustalW [16].  (Table 2) using the BLAST network service of NCBI (www.ncbi.nlm. gov/BLAST/). The analysis involved 28 nucleotide sequences. Ciliophoran parasite Ichthyophthirius multifiliis (U17354) was taken as an out-group. Phylogenetic cluster was based on the alignment of about 1800 bp long sequences. The phylogenetic tree was constructed using the Bayesian inference method implemented in the MrBayes program [v3.2.3] [17]. Four Markov Chain Monte Carlo (MCMC) chains were run for 10,000 generations, sampling every 10 generations. Less than 50% bootstrap replicates were collapsed. The DNA distance matrix (dnadist) was analyzed by Kimura 2parameter model [18] in MEGA6 [19].

RESULTS
Screening of 320 fish samples from different geographical areas of West Bengal, India yielded an Argulus infection rate of 10.63% (Table 1). Twelve out of 15 surveyed ponds had Argulus infection. The parasitic frequency index (PFI) was observed to be 4.65% in C. catla, 8.09% in L. rohita, 9.76% in C. mrigala, 5% in C. carpio and 20% each in H. molitrix and L. bata. The rate of infection was high in September (PFI: 17.02%) and October (PFI: 12.90%). The universal primer sets ERIB1 and ERIB10 successfully amplified approximately 1800 bp fragments of the 18S rRNA gene from Argulus spp. The PCR amplified products were sequenced, similarity calculations performed and compared the sequences of approximately 1.8 kb with sequences available in GenBank using BLAST network services and the results are presented in Tables 2 and 3.
The 18S rRNA sequences of five A. bengalensis and one A. siamensis of this study were submitted to the GenBank database by using Sequin software under nucleotide accession numbers KF583878, KF192316, KM016968, KM016969, KM016970 and KF583879, respectively. In the phylogenetic tree (Fig. 1) all the crustaceans were clustered together as a separate clade with two distinct lineages. In lineage-1, all Argulus spp. were clustered together, although with polytomy, giving many temporally based branches.  Values in parenthesis are the numbers of fish examined

DISCUSSION
Members of the genus Argulus spent major part of the life in surface and column waters and may have some predilection towards surface feeder, but enjoys both surface and column niches. Although 12 out of 15 surveyed ponds (80%) had Argulus infection, the infection rate of 10.63% in fish indicated a low prevalence. Contrarily, Paria and Konar [20] reported that 0.8-9.8% the ponds were affected out of the total 1332 freshwater fish ponds surveyed. According to them, Argulus was a not significant problem in West Bengal. The PFI was observed to be high in H. molitrix and L. bata followed by L. rohita, C. mrigala, C. carpio and C. catla. Contrary to this study, Argulus infection was reportedly more in Catla catla [21]; while Shella et al. [22] MBRC http://mbrc.shirazu.ac.ir 162 observed that L. rohita was susceptible to argulosis. The present study recorded high rate of Argulus infection in September (PFI: 17.02%) and October (PFI: 12.90%), i.e., the late rainy season. Earlier studies [23,24], however, recorded the highest Argulus infestation in winter and least in rainy season; while other studies [25,26] recorded incidence of argulosis throughout the year. Further, the fish from Haripota (PFI: 13.43%) and Bantala (PFI: 12.90%) had high Argulus infection than other locations, probably due to the presence of high organic loads, as the ponds from these localities use sewage water to raise fish. The infected fish exhibited a very few scattered signs of parasitic infection to low parasitic infection.   The objective of the present study was to characterize Indian Argulus spp. infecting the carps cultured in West Bengal, India by molecular tools as the 18S rDNA gene is the most conserved in nature and provide valuable information for comparative analysis [27]. In our study, the universal primer sets ERIB1 and ERIB10 successfully amplified approximately 1800 bp fragments of the 18S rRNA gene from Argulus spp. Earlier, there have been reports on the partial sequence of 28S rRNA of Argulus sp. [8], comparative phylogenetic analysis of Pentastomida and complete mitochondrial DNA (mt DNA) of A. americanus [10], molecular phylogeny of Branchiura, Pentastomida and other Maxillopoda based on mitochondrial 16S rRNA, nuclear 18S and 28S rRNA [28] and genetic variability within mtDNA regions of fish louse A. japonicus from Africa, Middle East and Asia [9]. These genetic data and phylo-geographical structure of a species provide the basic information required to understand their evolution and biogeographical history. According to Alam and Khan [29] populations having higher similarity are more homogeneous groups. The NCBI sequence similarity matrix between query and reference sequences of Argulus spp. showed genetic heterogeneity because the isolates were collected from different fish species and locations. This was also reflected in the phylogenetic tree (Fig. 1). Although all Argulus spp. were clustered together, polytomy was noticed possibly due to genetic heterogeneity. Similarly, Wadeh et al. [9] reported the genetic variability within fish louse A. japonicus from Africa, Middle East and Asia by examining sequence variability between cytochrome C oxidase subunit 1 (cox1) and NADH dehydrogenase subunits 1 and 4 (nad1 and nad4).
In a similar study, Sahoo et al. [11] reported the genetic variation of Argulus species collected from freshwater aquaculture systems of India by RAPD analysis. Their results indicated that the RAPD banding patterns obtained using host DNA were clearly distinct from that of parasites of all locations. They also constructed phylogenetic tree of Argulus species of India, but based on Nei's genetic distance and, hence, it is difficult to compare with their study. Both studies, however, recorded genetic heterogeneity among Argulus spp. collected from different fish species and locations. Yet, this is the first record of 18S rRNA gene sequence based molecular characterization of A. bengalensis and A. siamensis from India and their phylogenetic relationship with other Argulus spp. available in GenBank database. The molecular method appears to be a reliable diagnostic tool for Argulus characterization and can be useful for identifying Argulus spp. Our attempt to estimate genetic diversity among Indian Argulus spp., provided the baseline data for future work on population structure analysis of Argulus species, as very limited information exist for this economically important species, which would help identifying and managing these parasites in aquaculture.